For this manuscript, there were three types of methods performed to make our main conclusions: genetic diversity and structure analyses, downloading and mapping butternut fossil pollen during the last 20,000 years, and modeling and hindcasting butternut's (Juglans cinerea) distribution 20,000 years ago to present. Genetic analyses and species distribution modeling were performed in Emily Schumacher’s Github repository (https://github.com/ekschumacher/butternut) and pollen analyses and mapping were performed in Alissa Brown’s repository (https://github.com/alissab/juglans). Here is information detailing the Genetic data Data collection description: To perform genetic diversity and structure analyses on butternut, we used genetic data from the publication Hoban et al. (2010) and genetic data from newer sampling efforts on butternut from 2011 - 2015. Individuals were collected by Jeanne Romero-Severson, Sean Hoban, and Martin Williams over the course of ~ten years with a major sampling effort closer to 2009 followed up by another round of sampling 2012 - 2015. The initial 1,004 butternut individuals that were collected were genotyped by Sean Hoban and then the subsequent 757 individuals were genotyped in the Romero-Severson lab at Notre Dame non-consecutively. Genotyping was performed according to Hoban et al. (2008); DNA was extracted from fresh cut twigs using DNeasy Plant Mini kits (QIAGEN). PCR was performed by using 1.5 mM MgCl2, 1x PCR buffer [50 mm KCl, 10 mm Tris-HCl (pH 9.0), 0.1% Triton-X-100 (Fisher BioTech)], 0.2 mm dNTPs, 4 pm each forward and reverse primer, 4% Bovine Serum Albumin, 0.25 U TaKaRa Ex Taq Polymerase (Panvera), and 20 ng DNA template (10 μL total volume). The PCR temperature profile was as follows: 2 min at 94 °C; 30 cycles of 94 °C for 30 s, Ta for 30 s, and 72 °C for 30 s; 45 min at 60 °C; and 10 min at 72 °C on a PTC-225 Peltier Thermal Cycler (MJ Research). The process of assessing loci and rebinning for differences in years is detailed in the Schumacher et al. (2022) manuscript. Data files butternut_44pop.gen: Genepop file of original 1,761 butternut individuals, sampling described above, separated into original 44 sampling populations. butternut_24pop_nomd.gen: Genepop file of 1,635 butternut individuals, following rebinning based on researcher binning, reduced based on geographic isolation and missing data, organized into 24 populations. Used to generate all genetic diversity results. butternut_24pop_relate_red.gen: Genepop file of 993 butternut individuals, reduced for 25% relatedness, used to generate all clustering analyses. butternut_26pop_nomd.gen: Genepop file of 1,662 butternut individuals, reduced based on geographic isolation and missing data, including Quebec individuals, organized into 26 populations. Used to generate genetic diversity results with Quebec individuals. butternut_26pop_relate_red.gen: Genepop file of 1,015 butternut individuals, including Quebec individuals, reduced for 25% relatedness, used to generate clustering analyses with Quebec individuals. Fossil Pollen Data collection description: Pollen records for butternut were downloaded from Neotoma Paleoecology Database in 500-year time increments and visualized in 1,000 year-time increments 20,000 years ago to present. Data files butternut_pollen_data.csv: CSV of pollen records used for analyses and mapping. Includes original coordinates for each record (“og_long”, “og_lat”), the count of Juglans cinerea pollen at each site (“Juglans_cinerea_count”), and the age of the record (“Age”). To create the final maps, the coordinates were projected into Albers for each record (“Proj_Long,” “Proj_Lat”). Species Distribution Modeling and Hindcast Modeling Data collection description: We wanted to identify butternut's ecological preferences using boosted regression trees (BRT) and then hindcast distribution models into the past to identify migration pathways and locations of glacial refugia. Species distribution modeling was performed using boosted regression trees according to Elith et al. (2008). To run BRT, we needed to: 1. Reduce occurrence records to account for spatial autocorrelation, 2. Generate pseudo-absence points to identify the habitat where butternut is not found, 3. Obtain and extract the 19 bioclimatic variables at all points, 4. Select ecological variables least correlated with each other and most correlated with butternut presence. The BRT model that predicted butternut's ecological niche was then used to hypothesize butternut's suitable habitat and range shifts in the past. We downloaded occurrence records according to Beckman et al. (2019) as described here: https://github.com/MortonArb-ForestEcology/IMLS_CollectionsValue. The habitat suitability map generated from the BRT were projected into the past 20,000 years using Paleoclim variables (Brown et al., 2018). Data files butternut_BRT_var.csv: A CSV of the butternut presence and pseudoabsence points and extracted Bioclim variables (Fick & Hijman, 2017) used to run BRT in the final manuscript. Longitude and latitude coordinates are projected into Albers Equal Area Conic project, same with all of the ecological variables. Presence points are indicated with a 1 in the “PA” column and pseudo-absence points are indicated with a “0.” The variables most correlated with presence and least correlated with each other in this analysis were precipitation of the wettest month (“PwetM”), mean diurnal range (“MDR”), mean temperature of the driest quarter (“MTDQ”), mean temperature of the wettest quarter (“MTwetQ”), and seasonal precipitation (“precip_season”). References Brown, J. L., Hill, D. J., Dolan, A. M., Carnaval, A. C., & Haywood, A. M. (2018). PaleoClim, high spatial resolution paleoclimate surfaces for global land areas. Scientific Data, 5, 1-9 Elith, J., Leathwick, J. R., & Hastie, T. (2008). A working guide to boosted regression trees. Journal of Animal Ecology, 77, 802-813. Fick, S. E., & Hijmans, R. J. (2017). WorldClim 2: new 1‐km spatial resolution climate surfaces for global land areas. International Journal of Climatology, 37, 4302-4315. Hoban, S., Anderson, R., McCleary, T., Schlarbaum, S., and Romero-Severson, J. (2008). Thirteen nuclear microsatellite loci for butternut (Juglans cinerea L.). Molecular Ecology Resources, 8, 643-646. Hoban, S. M., Borkowski, D. S., Brosi, S. L., McCleary, T. S., Thompson, L. M., McLachlan, J. S., ... Romero-Severson, J. (2010). Range‐wide distribution of genetic diversity in the North American tree Juglans cinerea: A product of range shifts, not ecological marginality or recent population decline. Molecular Ecology, 19, 4876-4891.

Aim: Range shifts are a key process that determine species distributions and genetic patterns. A previous investigation reported that Juglans cinerea (butternut) has lower genetic diversity at higher latitudes, hypothesized to be the result of range shifts following the last glacial period. However, genetic patterns can also be impacted by modern ecogeographic conditions. Therefore, we re-investigate genetic patterns of butternut with additional northern population sampling, hindcasted species distribution models, and fossil pollen records to clarify the impact of glaciation on butternut. Location: Eastern North America Taxon: Juglans cinerea (L., Juglandaceae) (butternut) Methods: Using 11 microsatellites, we examined range-wide spatial patterns of genetic diversity metrics (allelic richness, heterozygosity, FST) for previously studied butternut individuals and an additional 757 samples. We constructed hindcast species distribution models and mapped fossil pollen records to evaluate habitat suitability and evidence of species’ presence throughout space and time. Results: Contrary to previous work on butternut, we found that genetic diversity increased with distance to range edge, and previous latitudinal clines in diversity were likely due to a few outlier populations. Populations in New Brunswick, Canada were genetically distinct from other populations. At the Last Glacial Maximum, pollen records demonstrate butternut likely persisted near the glacial margin, and hindcast species distribution models identified suitable habitat in the southern United States and near Nova Scotia. Main conclusions: Genetic patterns in butternut may be shaped by both glaciation and modern environmental conditions. Pollen records and hindcast species distribution models combined with genetic distinctiveness in New Brunswick suggest that butternut may have persisted in cryptic northern refugia. We suggest that thorough sampling across a species range and evaluating multiple lines of evidence are essential to understanding past species movements.

Data was cleaned and processed in R - genetic data cleaning and analyses and species distribution modeling methods were performed in Emily Schumacher's butternut repository and fossil pollen data cleaning and modeling was performed in Alissa Brown's juglans repository. Steps for performing data cleanining, analyses, and generating figures for the manuscript are described within each repo.